Serum calcium exists in two major forms. About half of the calcium in serum circulates as free ions and the other half is bound to circulating proteins. The small amount of calcium in blood is regulated by the parathyroid gland and the serum total protein level. Calcium ions participate in blood coagulation, neuromuscular conduction, maintenance of membrane function, intracellular regulations of glandular secretion, and control of skeletal and cardiac muscle contractility.
Colorimetric methods for determining the calcium content of serum must differentiate between spectrophotometric absorbance of a calcium-chromogen complex, such as calcium-arsenazo III, and spectrophotometric interferences due to lipemia, bilirubinemia, and hemolysis. Further, under alkaline pH conditions magnesium ion competitively binds to the chromogen, thereby producing assay interference. 8-Hydroxyquinoline effectively binds magnesium ion, thereby removing it as an interferant in the calcium assay.
Calcium reagents that include 8-hydroxyquinoline are disclosed in Neri et al., U.S. Pat. No. 4,448,889, Mezei et al., U.S. Pat. No. 4,382,122, Gindler, U.S. Pat. No. 3,754,865, and Stavropoulos et al., U.S. Pat. No. 3,938,954. However, these references do not disclose a single reagent for calcium assay. Rather, two reagents are required for the determination of calcium in serum. One reagent is an acidic dye reagent which includes a chromogen, a surfactant, and 8-hydroxyquinoline. The other reagent is a buffer reagent that includes an alkaline buffer.
A single calcium reagent is disclosed by Cleaver, U.S. Pat. No. 3,934,977. However, the reagent disclosed by Cleaver includes an acidic buffer. Further, reagent blank measurements, utilized in the calcium assay method disclosed in Cleaver, produce a high spectrophotometric absorbance.
Morin, U.S. Pat. No. 3,822,116, also discloses a single reagent for calcium assay. The reagent disclosed by Morin does not include 8-hydroxyquinoline.
Helger, U.S. Pat. No. 3,798,000, discloses a single reagent for calcium assay. The reagent has an alkaline pH and includes either 8-hydroxyquinoline sulfate or 8-hydroxyquinoline.
Bates et al., European Patent Application No. 88113754.1, discloses a single reagent for calcium assay. The reagent includes arsenazo III, but utilizes n acidic buffer and does not include 8-hydroxyquinoline.
Baginski et al., "Direct Microdetermination of Serum Calcium," Clin. Chim. Acta. 46, 49-54 (1973), discloses a multiple reagent system for calcium determination (p. 50). One of the reagents includes 8-hydroxyquinoline, dimethylsulfoxide, and acid (concentrated hydrochloric acid) (p. 50).